Transfection of brain capillary endothelial cells in primary culture with defined blood–brain barrier properties

Abstract

BackgroundPrimary brain capillary endothelial cells (BCECs) are a promising tool to study the blood–brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells’ integrity.MethodsPrimary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.ResultsThe cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.ConclusionsThe data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Effects of electromagnetic pulse exposure on gelatinase of blood–brain barrierin vitro

“…The rats were killed with rapid decapitation (Burkhart et al, 2015). After careful removal of the meninges, the cerebral cortex was separated mechanically from the brain stem in ice-cold D-Hanks solution and chopped into pieces less than 1 mm 3 on a side.…”

The vascular basement membrane in the healthy and pathological brain

“…154,155 A strategy to further allow such growth factors to enter the brain is to transfect BCECs with cDNA injected into the circulation. [156][157][158] Thus, the binding capacity of the vascular basement membrane must be taken into consideration when designing delivery strategies across the BBB, as the growth factors could get trapped there.…”

Meningitic Escherichia coli-induced upregulation of PDGF-B and ICAM-1 aggravates blood-brain barrier disruption and neuroinflammatory response

“…The blood-brain barrier (BBB) consists of brain microvascular endothelial cells (BMECs), pericytes, and astrocytes and separates the brain parenchyma from blood circulation. The BBB ensures the brain's energy supply and stabilizes the microenvironment by discharging CNS metabolites and neurotoxic agents into circulation, while preventing circulating neurotoxic agents, inflammatory factors, or immune cells entering the CNS [7]. BMECs are the most direct and functional structural component of the BBB and are closely linked by the expression of tight junction (TJ) proteins, including Claudins, Occludin, and cytoplasmic zonala-occludin family members (such as ZO-1, ZO-2, and ZO-3) [8].…”